Withania somnifera (L.) Dunal ameliorates neurodegeneration and cognitive impairments associated with systemic inflammation
Systemic inflammation driven neuroinflammation is an event which correlates with pathogenesis of several neurodegenerative diseases. Therefore, targeting peripheral and central inflammation simultaneously could be a promising approach for the management of these diseases. Nowadays, herbal medicines are emerging as potent therapeutics against various brain pathologies. Therefore, in this contemporary study, the neuroprotective activity of Ashwagandha (Withania somnifera) was elucidated against the inflammation associated neurodegeneration and cognitive impairments induced by systemic LPS administration using in vivo rat model system.
To achieve this aim, young adult wistar strain male albino rats were randomized into four groups: (i) Control, (ii) LPS alone, (iii) LPS + ASH-WEX, (iv) ASH-WEX alone. Post regimen, the animals were subjected to Rotarod, Narrow Beam Walking and Novel Object Recognition test to analyze their neuromuscular coordination, working memory and learning functions. The rats were then sacrificed to isolate the brain regions and expression of proteins associated with synaptic plasticity and cell survival was studied using Western blotting and Quantitative real time PCR. Further, neuroprotective potential of ASH-WEX and its active fraction (FIV) against inflammatory neurodegeneration was studied and validated using in vitro model system of microglial conditioned medium–treated neuronal cultures and microglial-neuronal co-cultures.
Orally administered ASH-WEX significantly suppressed the cognitive and motor-coordination impairments in rats. On the molecular basis, ASH-WEX supplementation also regulated the expression of various proteins involved in synaptic plasticity and neuronal cell survival. Since microglial-neuronal crosstalk is crucial for maintaining CNS homeostasis, the current study was further extended to ascertain whether LPS–mediated microglial activation caused damage to neurons via direct cell to cell contact or through secretion of inflammatory mediators. ASH-WEX and FIV pretreatment was found to restore neurite outgrowth and protect neurons from apoptotic cell death caused by LPS-induced neuroinflammation in both activated microglial conditioned medium–treated neuronal cultures as well as microglial-neuronal co-cultures.
This extensive study using in vivo and in vitro model systems provides first ever pre-clinical evidence that ASH-WEX can be used as a promising natural therapeutic remedial for the prevention of neurodegeneration and cognitive impairments associated with peripheral inflammation and neuroinflammation.
KeywordsAshwagandha Lipopolysaccharide Microglia Neuroinflammation Neuroprotection Synaptic plasticity
Ashwagandha leaf water extract
BCL2 associated death promoter
B-cell lymphoma 2
B-cell lymphoma 2 extra large
Brain derived neurotrophic factor
Active chloroform fraction (Fraction IV)
Growth associated protein 43
Glyceraldehyde 3-phosphate dehydrogenase
High Fat Diet
Ionized calcium-binding Adaptor Molecule 1
Integrated density values
Inducible nitric oxide synthase
inositol 1,4,5- triphosphate
Microtubule–associated protein 2
Mitogen activated protein kinase
Neural Cell adhesion molecule
Nuclear factor kappa light-chain-enhancer of activated B cells
- NOR Test
Novel Object Recognition Test
- PI score
Preference index score
Phosphoinositide phospholipase C gamma
Polysialylated-neural cell adhesion molecule
Post synaptic density protein 95
Reactive nitrogen species
Reactive oxygen species
Tumor necrosis factor alpha
Tropomyosin receptor kinase B
Neuroinflammation is considered to be a critical risk factor responsible for impaired neurogenesis, synaptic plasticity and cognitive deficits associated with several neurodegenerative diseases (NDDs) . The prevalence of the neurodegenerative diseases (NDDs) has been increasing significantly with the concurrent increase in the life expectancy globally. Although brain is immunologically privileged, various epidemiological studies have positively correlated the systemic inflammation linked with various chronic inflammatory diseases like T2D, atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, Crohn’s disease, ulcerative colitis etc. with the AD and PD risk [2, 3]. Systemic inflammatory responses signal to the brain through neural route via terminals of vagus nerve and humoral route via circumventricular organs lacking BBB. Inflammatory signals also communicate across the BBB via interactions between the perivascular macrophages and resident microglia through activation of endothelial cells [2, 4]. The microglia, brain immune cells along with glia and neurons are found to be implicated in neuroinflammation linked to pathology of NDDs . They are exquisitely sensitive to endogenous (like stroke, autoimmune processes) and exogenous (e.g. pathogens or psychological stressors) challenges and undergoes phenotypic switch to adopt an activated neurotoxic state. This leads to perturbances in healthy neuron-glial interactions and result in impaired neuronal plasticity and cognitive functions .
Activated microglia can induce significant and highly destructive neurotoxic effects directly or indirectly by excessive production of a large array of neurotoxicity mediators which act alone or in combination to damage neurons [7, 8]. Normally microglial cells are neuroprotective i.e. they safeguard the neurons by killing pathogens, removing debris, amyloid β and also provide them with nutritive factors such as BDNF and IGF1. But while attempting to limit infection or when this protective function is not fully accomplished, they accidentally damage the neurons [9, 10]. Co-activation of pro–inflammatory markers and associated cytotoxic products during neuroinflammation are detrimental for neurons by modulating expression of synaptic proteins and their functions. Postmortem brains from bipolar disorder (BD) and Schizophrenic patients showed upregulated neuroinflammatory markers along with decreased synaptic markers (drebin and synaptophysin) and have been linked with cognitive impairments in BD and SZ patients . Microglia mediated neuroinflammation also negatively impacts the neurogenesis by causing impaired neuronal survival and proliferation of the new neurons. Ekdahl et al. (2003) demonstrated that LPS from E.coli administered intracortically or intraperitoneally induces strong immune responses in brain and reduces both neurogenesis and neurodifferentiation . The consequences of inflammation on neurogenesis negatively impacts cognition and spatial information . Neuroinflammation also affects the recruitment and correct integration of newborn neurons into hippocampal network encoding spatial information . The current inputs from literature suggest that simultaneously targeting peripheral and central inflammation may be a promising strategy to combat neurodegeneration.
Withania somnifera (L.) Dunal (Ashwagandha) has been acknowledged as an adaptogen or rejuvenator in the Ayurvedic medicine from centuries for its potency to normalize physiological functions via regulating the endocrine and immune functions [14, 15]. “Withanolides” inclusive of steroidal alkaloids and lactones are the major pharmacologically active constituents of Ashwagandha extracts prepared from leaves, shoots and roots of plant. Out of these, withaferin A, withanone, withanolide A and withanolide D constitute the major fractions. Withaferin A has been suggested as a natural anti-inflammatory and neuroprotective candidate in various neurological disorders model like spinal cord injury, high-fat diet-induced obesity and several NDDs models [16, 17, 18]. The neurite outgrowth promoting activity of the Ashwagandha root methanol extract has been attributed to presence of Withanolide A, withanoside IV, and withanoside VI as the active components [19, 20]. All these active ingredients collectively contribute to neuroprotective activity of Ashwagandha. Majority of the studies on Ashwagandha reported previously in literature have evaluated its neuroprotective activities using alcoholic formulations of roots [21, 22, 23, 24, 25, 26]. In this study, we used the leaf based water extract of Ashwagandha to scientifically ascertain the doctrinal use of this Ayurvedic elite, a pharmacological important plant. Moreover, aqueous formulations are easy to prepare and safe, as it avoids the use of organic solvents and scarification of plant is not required.
Ashwagandha leaf water extract (ASH-WEX) and its active fraction (FIV) were previously reported by us to prevent the LPS-induced activation of microglial cells and inhibited the secretion of various pro-inflammatory mediators in primary microglial cultures and BV-2 cell line used as in vitro model system . The anti-neuroinflammatory activity of ASH-WEX and FIV has been ascribed to presence of withaferin A and withanone as revealed by HPLC analysis. Moreover, ASH-WEX was found to prevent systemic lipopolysaccharide (LPS) induced reactive gliosis, expression of pro-inflammatory cytokines and nitro-oxidative stress products. Additionally, NFκB, P38 and JNK MAPKs pathways were found to be the principal molecular mechanism underlies the anti-inflammatory activity of ASH-WEX . Therefore, the objective of current study was to further explore the neuroprotective potential of ASH-WEX to ameliorate cognitive impairments and synaptic plasticity changes induced by systemic LPS associated inflammation using both in vitro and in vivo model system. LPS-induced neuroinflammation has been reported to induce CNS-mediated behavioral responses collectively known as “sickness behavior” which are defined by impaired motor functions, diminished environmental or social exploration along with learning and memory dysfunctions . Therefore, systemic LPS-induced inflammation has been often used in literature to understand molecular mechanisms of cognitive dysfunctions due to inflammation and also to develop targeted remedies for neurological symptoms [3, 30].
The beneficial effect of the ASH-WEX against systemic LPS-induced cognitive deficits was assessed by behavioral tests such as Narrow beam walking test and Novel object recognition (NOR) test and motor coordination by Rotarod test. Further to elucidate the molecular basis of behavioral effects, the expression of various proteins regulating synaptic plasticity and cell survival were studied. Further to investigate the role of microglia in inflammation driven neurotoxicity and neuroprotective activity of ASH-WEX, we used in vitro model system of conditioned media and co-culture regimen. The current study manifestated that ASH-WEX may be potential candidate to ameliorate neurodegeneration and behavioral deficits of learning and memory as well as motor coordination caused by peripheral inflammation.
Animals and experimental conditions
3–4 months old disease free wistar strain male albino rats with body weight ranges from 130 to 150 g were purchased from NIPER, Mohali, Punjab, India and used in the present study. 3–4 animals were housed per cage with free access to food and water under controlled temperature (25 ± 2 °C) and humidity conditions with 12 h light/dark cycle. Protocols were duly approved by Institutional Animal Ethical Committee (IAEC) of the Guru Nanak Dev University, Amritsar, Punjab, India as per Committee for the Purpose of Control and Supervision of Experiments on Animals (CPSCEA) guidelines for care and use of laboratory animals laid down by same committee. All experimental procedures were conducted according to ARRIVE guidelines.
Control: Orally administered with water as vehicle for 8 weeks
LPS alone group: Orally administered with water (4 ml/kg body weight) as vehicle for 8 weeks and were injected intraperitoneally with single dose of LPS (5 mg/kg) on last day of treatment regimen.
LPS + ASH-WEX group: animals of this group were orally administered with ASH-WEX (4 ml/kg body weight) for 8 weeks and injected with single i.p. dose of LPS (5 mg/kg) on last day.
ASH-WEX alone: orally administered with only ASH-WEX (4 ml/kg body weight) for 8 weeks.
12 h after LPS treatment, animals were assessed for their behavior using various behavioral tests like Rotarod, Narrow beam walking test, Novel object recognition test. For detailed biochemical and molecular studies, animals were anesthetized by injecting thiopentone intraperitoneally at the dosage of 1 unit/10 g and decapitated after cervical dislocation to isolate brains. Further, the brains were microdissected to isolate the hippocampus and pyriform cortex (PC) regions of brain.
Animals were given ASH-WEX orally at the dose of the 4 ml/kg/day extract (equivalent to 140 mg/kg/day dry weight of ASH-WEX) standardized and reported previously by our lab [28, 31, 32, 33]. For in vitro studies, ASH-WEX and one of its active fraction (FIV) were used at the concentration of 0.2% and 10 μg/mL, respectively as reported in our previous study .
Rotarod test was performed to monitor the motor coordination of the animals (n = 8/group). Its apparatus is an automated motor operated treadmill (Rotamex-5; Columbus Instruments). It consists of rotating spindle having dimensions 7.0*9.5 cm for rats with 44.5 cm fall height from the rod center. This test was carried out at a constant speed of 10 rpm on individual animal for 300 s and was recorded for the time spent by the animals on the rotating rod and number of falls from it.
Narrow beam walk test
Narrow beam walk test was carried out to evaluate the motor balance and grip strength of the rats (n = 8/group) as described by Goldstein and Davis (1990) . The apparatus is equipped with 1 m long and 1–2 cm wide beam placed horizontally at 30 cm elevation above the floor. Rats were allowed to walk across the beam from one end to dark home cage at the other end. The test consists of 2 days training and 1 day testing. The frequency of paw slippage and time taken to cross the beam were recorded. The score was represented as the cumulative of two successful trials on test day and averaged for each animal in a group.
Novel object recognition (NOR) test
The NOR test was performed to measure object recognition working memory of animals (n = 8/ group) according to protocol as described earlier . Briefly, the test consists of 5 mins empty box habituation of animals for 2 days followed by their 5 mins familiarization with two objects for 3 consecutive days as training program. On the test day, the least preferred object was replaced by novel one and animals were allowed to explore and then assessed for their preference for the objects, exploration time and number of episodes. Further the animals were analyzed for grooming and the rearing behavior in the open field box of NOR test to correlate the effect of stress and anxiety caused by LPS-induced neuroinflammation on memory and cognition. Any grooming bout of duration more than 5 secs was considered as actual grooming bout. The number of grooming episodes and total time spent in grooming were recorded and averaged for each group.
Western blotting was performed according to protocol as described earlier (Gupta and Kaur 2018) . Briefly the animals (n = 4–5/group) were anesthetized by injecting thiopentone intraperitoneally at the dosage of 1 unit/10 g and decapitated after cervical dislocation to isolate brains. Further, the hippocampus and pyriform cortex (PC) regions of brain were microdissected, homogenized and their protein lysates were prepared. Protein concentration was estimated using Bradford method followed by preparation of samples which were then electrophoretically resolved and transferred onto the membranes. After blocking the membranes, they were probed with monoclonal anti-PSA-NCAM (1:1500), anti-NCAM (1:2000), anti-CaMKIIα (1:2000), anti-CaN (1:2000) anti-phospho Akt (1:2000), anti-MAP2 (1:2000), anti-GAP43 (1:2500), (Sigma-Aldrich St. Louis, MO, USA), anti-TrkB (1:1000) (Trk A & B antibody sampler kit, Cell Signaling Technology, MA, USA), anti-Bcl-xL (1: 1500) for overnight at 4 °C. Further, the membranes were washed and incubated with their respective secondary antibodies (Genei) HRP-labeled anti-mouse IgM (1:2000) for PSA-NCAM, anti-rabbit IgG (1:5000) for pAkt1, anti-rabbit IgG (1:1500) for anti-TrkB, anti-mouse IgG (1:5000) for CaMKIIα, CaN, MAP2, GAP43 (Merck Millipore, USA) for 2 h at 25 °C. α-Tubulin was used as internal control for protein loading. ECL Prime Western Blotting Detection Reagent was used for visualizing immunoreactive bands using Image Quant LAS 4000 system (GE Healthcare, Little Chalfont, UK). For western blots analysis, the expression of each control (in triplicates) was taken as 100% and then percentage change in expression of protein of interest in test samples compared to corresponding control was calculated. The average and corresponding standard error was then calculated using MS Excel and Sigma Stat. Standard error bars were also plotted on the scale of 100. This way of western blot analysis is well accepted and reliable one. Researchers usually present the relative expression in their test samples either in ratio of control (at the scale of 1) [36, 37, 38] or percent of control (at the scale of 100) [39, 40, 41, 42, 43]. We have been presenting our western data in previous lab publications in this pattern [27, 28, 32, 33, 44]. Data was analyzed for change in protein expression.
Quantitative real time PCR (RT-PCR) for mRNA expression analysis
mRNA expression analysis was done according to protocol as described earlier . Briefly, the total RNA was extracted from brain tissues using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer’s instructions (n = 4–5/group). Further, the RNA was reverse transcribed using M-MLV reverse transcriptase to prepare the cDNA. Amplification of gene of interest was then done from 50 μg of cDNA using StepOne Plus Real Time PCR system (Applied Biosystems, Foster City, CA, USA) according to SYBR chemistry. GAPDH was used as an internal control. The ‘Livak method’ was applied to calculate the relative gene expression and data was presented as 2 − ΔΔCt ± SEM.
Primary microglial cultures
Microglial enriched cultures were obtained from primary mixed glial cultures from 1-day-old pups using mild trypsinization method  with slight modification as described earlier . Isolated microglial cells were seeded in multiwell plates by trypsinization with 0.25% trypsin-EDTA for 10 mins and treated after 24 h of seeding. For preparation of the microglial conditioned medium, primary microglial cells were pretreated with 0.2% and 10 μg/mL of ASH-WEX and FIV, respectively for 2 h prior to activation with 100 ng/mL LPS [LPS (rough strains) from E. coli F583 (Rd mutant)]. The microglial conditioned medium was harvested after 36 h of treatment, centrifuged to make cell free and used freshly for different experiments. Conditioned medium from different groups were designated as CM-Control, CM-ASH-WEX, CM-FIV, CM-LPS, CM-LPS + ASH-WEX, CM-LPS + FIV.
Primary neuronal cultures
Primary neuronal cultures were prepared from 1-day-old (cortical), 2-day-old (hippocampal) and 7-day-old (cerebellar) pups, respectively using the procedure followed by Frandsen and Schousboe (1990) with some modifications . Briefly, the pup brain was dissected to collect cortex, hippocampus and cerebellum regions; their meninges were carefully removed and the tissue was digested with 0.025% (w/v) trypsin-EDTA solution containing 0.1% DNase for 15 mins at 37 °C. Activity of trypsin was stopped by adding equal amount of trypsin inhibitor solution or media and centrifuged (1000 rpm, 4 mins). The tissue was titurated and the cells were resuspended in the Neurobasal medium. Then the cells were seeded on poly-L-lysine coated 24 well culture plates and maintained in Neurobasal medium (supplemented with bFGF and B12 supplement) at 37 °C in 5% humidified atmosphere.
Primary microglial-neuronal co-culture
For primary microglial–cerebellar neurons co-culture, firstly the primary microglia cultures were prepared from 1-day-old pups as described above. Microglial cells obtained were seeded at a density of 15,000 cells/mL on PLL coated coverslips. After 24 h, microglial cells were pretreated with ASH-WEX and FIV for another 24 h. Then ASH-WEX and FIV containing medium was removed and cerebellar neurons were cultured from 7-day-old pups as detailed above and seeded on the top of the microglial monolayer in a ratio of 2:1 (neuronal: microglial ratio). After 4 h of seeding cerebellar neurons, microglial-neuronal cultures were challenged with 100 ng/ml LPS for 36 h. Microglial-neuronal co-cultures were then used for MAP2 immunostaining and DAPI nuclear staining to determine the percentage apoptotic cell death. Images captured for MAP2 immunostaining were used to determine the percentage neurite outgrowth. Experiments were performed in triplicate. Neurite lengths were determined from at least 100 cells per group using Image Pro Plus software (Media Cybernetics, Silver Spring, USA) and then analysis was done using Microsoft excel and Sigma Stat for windows (version 3.5).
The control and treated cells were washed with 1X PBS thrice, fixed with 1:1 acetone and methanol followed by permeabilization with 0.3% Triton X-100 in PBS (PBST). After blocking with 2% BSA in PBS, cells were incubated with anti-MAP2 (1:200, Sigma-Aldrich) monoclonal antibody diluted in blocking solution for 24 h at 4 °C in a moist chamber. Then the cells were washed twice or thrice with 0.1% PBST followed by incubation with anti-mouse IgG 488 secondary antibody (diluted at a concentration of 1:500 in 2% BSA) for 2 h at room temperature. For nuclear staining, cells were incubated with 1:5000 dilution of DAPI in 1X PBS for 10 mins. Cells were then mounted with anti-fading reagent (Fluoromount, Sigma-Aldrich). Nikon AIR Confocal Laser Microscope was used for capturing the images. Experiments were performed in triplicate. For quantifying MAP2 relative optical intensity, ROIs were selected from different cells (at least 100 cells per group) and analyzed for the intensity using NIS elements AR analysis software version 4.11.00. For neurite outgrowth analysis, at least 100 cells per group were measured for length of neurites using the Image Pro Plus software (Media Cybernetics, Silver Spring, USA) and percentage neurite outgrowth per group was calculated using Microsoft excel and Sigma Stat for windows (version 3.5).
The mean ± SEM of values calculated from minimum of three independent experiments were used to express data. Student’s t-test, one-way ANOVA (Holm-Sidak post hoc method) and two-way ANOVA have been employed to assess the level of significance by using Sigma Stat software for Windows (version 3.5). Values showing p ≤ 0.05 were acceptable as statistically significant. Only where the significance level comes ≤ 0.05 while comparing different groups, the symbol representing significance has been entered in the graphs.
ASH-WEX pretreatment improved neuromuscular coordination
ASH-WEX suppressed LPS-induced cognitive impairments
Moreover, LPS alone-treated rats exhibited longer latency to start grooming with reduced number of grooming bouts and spent less time in grooming during NOR test. Impaired grooming behavior probably could be due to sickness induced by LPS (Fig. 2d and e; p ≤ 0.005). LPS + ASH-WEX- and ASH-WEX alone-treated rats exhibited normal grooming behavior with more duration and frequency of grooming bouts comparable to control rats. Similarly, LPS-treated animals exhibited less number of rearing than control group animals, while, LPS + ASH-WEX- and ASH-WEX alone-treated animals exhibited maximum number of rearing implying their interest in exploration of empty box and reduction in LPS-induced stress by ASH-WEX administration (Fig. 2f; p ≤ 0.001).
ASH-WEX and the expression of synaptic plasticity and neuronal survival proteins
Further, the expression of Ca2+ and calmodulin dependent serine threonine protein phosphatase (calcineurin) and protein kinase (CAMKIIα) were studied in hippocampus and PC regions of brain which play an integral role in synaptic transmission modulation. LPS significantly reduced CAMKIIα expression (up to 71%) as compared to control rats (taken as 100%) while its expression was restored up to 86% in LPS + ASH-WEX-treated rats compared to LPS alone group in hippocampus region (Fig. 3d and e; p ≤ 0.05). In PC region, no significant variations were found in CAMKIIα expression amidst all the four group of animals (Fig. 3d and f; p ≤ 0.05). Its expression remained normal in both brain regions of ASH-WEX alone-treated animals. In contrast, the expression of calcineurin was not altered among all groups in hippocampus (Fig. 3d and e) and PC (Fig. 3d and f) regions of brain.
ASH-WEX and BDNF-TrkB receptor/ PI3K-Akt pathway
Akt further binds to BAD protein (BCL2 associated death promoter) and causes its inactivation. BAD protein complexes with 14-3-3 proteins in cytoplasm which prevents association with mitochondrially localized Bcl-xL and therefore inhibits apoptosis. LPS treatment marginally reduced Bcl-xL expression up to 95 and 82% from hippocampus and PC regions, respectively in comparison to control rats (taken as 100%), whereas, ASH-WEX administration restored the expression of Bcl-xL up to 107 and 99% in hippocampus and PC regions, respectively (Fig. 4a-c; p ≤ 0.05). ASH-WEX alone-treated rats exhibited near control level expression of Bcl-xL in both brain regions. mRNA expression of Bcl-xL was observed to be near control level in LPS + ASH-WEX- and ASH-WEX alone-treated animals but was significantly reduced with LPS treatment from hippocampus region (Fig. 4d; p ≤ 0.05). In PC region, no significant variations were found in mRNA levels of Bcl-xL among all the groups (Fig. 4e; p ≤ 0.05).
BDNF-TrkB pathway also activates PLCγ signaling pathway which further mediates the signals through IP3 by binding to its receptor and causing rapid release of Ca2+ involved to regulate neuronal activity and synaptic transmission. In PC region, ASH-WEX treatment showed 2-fold increase in expression of PLCγ which was decreased significantly in LPS alone-treated animals (Fig. 4e; p ≤ 0.05). mRNA expression of PLCγ remained unaffected among all groups in hippocampus region (Fig. 4d). LPS treatment significantly reduced the mRNA levels of IP3R gene in both hippocampus and PC regions, whereas, its expression was significantly upregulated by ASH-WEX administration (Fig. 4d and e; p ≤ 0.05).
ASH-WEX and FIV protected neurons against microglial mediated neurotoxicity
To further validate the neuroprotective effect of ASH-WEX and FIV against LPS-induced neuroinflammation, cell free conditioned medium (CM) of microglial cultures was prepared and tested (as per details in material and methods section). Direct treatment of LPS at the concentration of 100 ng/mL was found to be non toxic to neurons, whereas, neuronal cultures treated with CM-LPS showed significant downregulation in expression of MAP2 (Fig. 5a-c column II) as compared to CM-Control (Fig. 5a-c column II) and LPS alone-treated neurons (Fig. 5a-c column I). In contrast, treatment of neuronal cultures with CM-LPS + ASH-WEX and CM-LPS + FIV (Fig. 5a-c column II) showed near normal expression of MAP2 as compared to LPS + ASH-WEX- and LPS + FIV-treated neurons (Fig. 5a-c column I) as depicted by MAP2 immunostaining (Fig. 5a-c) and their intensity plots (Fig. 5d-f; p ≤ 0.05). MAP2 expression remained near control level in neurons treated with CM-ASH-WEX, CM-FIV, ASH-WEX and FIV alone. Since, neuroinflammation impairs the axonal regrowth and contributes to lack of neural regeneration, so confocal images of MAP2 immunostaining were further analysed for percentage neurite outgrowth (Fig. 5g-i). CM-LPS treatment group showed significant degeneration of neurite outgrowth as compared to CM-Control- and LPS alone-treated cortical, hippocampal and cerebellar neuronal cultures (Fig. 5g-i; p ≤ 0.05). CM-LPS + ASH-WEX and CM-LPS + FIV treatment also restored the neurite outgrowth as shown in histograms (Fig. 5 g-i).
ASH-WEX and FIV prevented the neuronal apoptosis against microglial mediated inflammatory injury
Apoptosis was determined by analyzing nuclear morphology using 4′,6′-diamidino-2-phenylindole (DAPI) staining (Fig. 6). 4, 6-Diamidino-2-phenylindole (DAPI) is a DNA binding dye and is commonly used assay for detection of apoptosis in cells. Apoptotic cells have more permeability for dye so these cells show more intense blue fluorescence due to chromosomal condensation, whereas, normal cells show uniformly stained round nucleus with clear margins. So DAPI staining can be used to observe differences between apoptotic and non-apoptotic cells based on the intensity of fluorescence and the conformation of nucleus. Neurons treated with CM-LPS for 24 h showed both disintegration and condensation of nuclei resembling apoptotic cell death (Fig. 6a-c) and caused significant apoptosis of neurons as indicated by highest percentage of apoptotic cells (Fig. 6d-f; p ≤ 0.05). CM-LPS + ASH-WEX and CM-LPS + FIV treatment effectively suppressed apoptosis of neurons. CM-ASH-WEX, CM-FIV, ASH-WEX and FIV alone treatment did not affect the survival of the neurons. However, CM-LPS + ASH-WEX and CM-LPS + FIV treatment restored the neuronal cell viability thus suggesting the neuroprotective effect of ASH-WEX and FIV (Fig. 6).
ASH-WEX and FIV also protected the neurons in microglial-neuronal co-culture
To further investigate whether microglia directly participate in neurotoxicity via cell to cell contact or only through secreted chemical mediators of inflammation, we performed the primary microglia-cerebellar neurons co-culture and analyzed for MAP2 neuronal protein expression, percentage neurite outgrowth and percentage neuronal apoptotic cell death (Fig. 7). Microglial-neuronal co-cultures treated with LPS alone exhibited less number of MAP2 positive neurons as well as (up to 67%) reduced MAP2 expression (Fig. 7c; p ≤ 0.05) and showed neuronal degeneration with retracted neurite outgrowth (Fig. 7b) as compared to control co-cultures. Further, the proportion of the cerebellar neurons showing apoptosis like nuclear condensation reached up to 66% (Fig. 7d; p ≤ 0.05) of the total neuronal population after 36 h of co-culture. In contrast, ASH-WEX and FIV treatment normalized MAP2 expression, sustained neuronal viability with increase in length of neurite outgrowth (31% for LPS + ASH-WEX and 52% for LPS + FIV) and reduced the percentage of apoptotic neurons up to 15% (ASH-WEX) and 11% (FIV) as compared to LPS alone-treated co-cultures (Fig. 7b-d; p ≤ 0.05).
Peripheral inflammatory responses elicited by single i.p. dose of LPS were observed to result in several behavioral deficits in the animals. Diminished social and environmental exploration, poor learning and memory and impaired neuromuscular coordination etc. were observed in these animals which are collectively termed as LPS-induced sickness behavior . During locomotor coordination test, LPS-treated rats were lethargic as discerned by maximum number of falls and less amount of time spent on the rotating rod, whereas, animals orally fed with ASH-WEX before LPS treatment performed well on the rotating rod with less number of falls indicating normal neuromuscular coordination (Fig. 1a-b). Similarly in Narrow beam walking test, LPS-treated rats took longer time to cross the beam with poor balance and higher frequency of paw slippages while ASH-WEX supplemented rats transverse the beam in shorter span of time like control rats (Fig. 1c-d). Further, during NOR test, LPS-treated animals preferred old object over the novel one (PI ≤ 0.5) with more frequency of episodes and time spent in exploring old object, whereas, ASH-WEX pretreated group highly preferred the novel object (PI ≥ 0.5) with more time spent in exploration of novel object (Fig. 2a-c). These observations from NOR test suggest ASH-WEX improved memory and learning which got impaired due to sickness behavior associated with LPS-induced inflammatory responses.
Healthy animals exhibit spontaneous self paced grooming defined by a stereotyped cephalocaudal sequence [48, 49]. Conversely animals with high stress and anxiety levels show interrupted and disorganized grooming pattern. LPS injected rats showed anxious behavior along with less number and duration of grooming bouts with disrupted and disorganized pattern which may be attributed to sickness behavior induced by LPS. This finding was consistent with other studies reporting that i.p. injection of LPS caused the reduction in grooming bouts and fur cleaning numeric score in inbred mice . ASH-WEX supplementation attenuated anxiety-like behavior and improved frequency and duration of grooming bouts with organized and structured pattern in these animals, suggesting anxiolytic activity of Ashwagandha (Fig. 2d-e).
These observations were also supported by changes in rearing activity which is an indices of exploratory behavior found in rodents . LPS + ASH-WEX- and ASH-WEX alone-treated rats showed maximum number of rearing episodes compared to LPS-treated rats indicative of improved exploratory behavior (Fig. 2f). Various experimental and clinical studies have reported memory enhancing and learning improvement potential of alcoholic and water based formulation of Ashwagandha . Recently our lab reported that aqueous extract of Ashwagandha has immunomodulatory and anxiolytic effects and improved memory retention and learning ability by alleviating cellular stress via regulating synaptic plasticity and cell survival signaling cascades in acute sleep deprived and HFD fed obese rats [32, 33, 35, 53].
Different brain regions have differential susceptibility to LPS-induced neuroinflammation with hippocampus and pyriform cortex (PC) being highly affected areas. Since hippocampus region is well known for its role in synaptic plasticity, regulation of spatial and learning memory, so expression of synaptic plasticity and neuronal survival protein markers were studied in hippocampus and PC regions. Further detailed study of molecular mechanisms revealed that LPS significantly upregulated the expression of 140 and 180 kDa isoforms of NCAM along with PSA-NCAM in hippocampus and PC regions. However, ASH-WEX pretreatment normalized the inflammation induced increase in PSA-NCAM and NCAM expression and seems to play important role in facilitating memory consolidation and regulating synaptic plasticity (Fig. 3). Both PSA-NCAM and NCAM expression is reported to be upregulated under various stress conditions and mood disorders [54, 55]. Enhanced expression of both NCAM and PSA-NCAM in LPS-treated rats may be the compensatory neuroprotective mechanism in stress vulnerable neuronal circuits to combat stressful conditions. They may be instrumental to maintain neural networking since repair and functional recovery after brain injury critically depends on structural and functional plasticity of preserved neuronal networks. Menzel et al. (2016) reported that antibody blocking of neural cell adhesion molecule L1 inhibits activated immune T cell adhesion and attack to neurons in co-cultures thereby reduces the neurodegeneration . The study suggested that downregulation of L1 is an adaptive endeavor to promote neuronal self defense in response to neuroinflammation and mitigates inflammatory neuronal injury.
LPS administration to animals downregulated CaMKIIα protein expression in hippocampus region, while its expression remained unaffected in PC region of brain. Interestingly ASH-WEX supplementation to LPS-treated rats enhanced CaMKIIα protein expression. On the other hand, expression of calcineurin was found to be unaffected in all the groups in both regions of brain (Fig. 3). Functionally, calcium and calmodulin serine/ threonine protein kinases CaMKII and protein phosphatases calcineurin coordinately regulate the calcium dependent synaptic plasticity including LTP. They also play key role in release of neurotransmitters, learning and memory consolidation . Decrease in expression of CaMKIIα in LPS-treated rats may be associated with performance deficits as observed in NOR test. Zhang et al. (2015) also reported that inflammatory trigger Aβ, elevated the Ca2+ levels and reduced CaMKII activity leading to degeneration of APPKI (amyloid precursor protein knock in) neurons along with impairment of calcium dependent synaptic plasticity and memory loss in AD . ASH-WEX pretreated group showed attenuation of cognitive deficits, promoted structural and functional integrity as well as improved working memory and learning functions thus suggesting their potential role against inflammation mediated neurodegeneration.
Accumulating evidence support that BDNF-TrkB is a major pathway implicated in the plethora of brain functions including neuronal cell survival, neurite growth, cell migration, synapse formation etc. [61, 62]. LPS treatment lead to reduction in mRNA expression of BDNF and its TrkB receptor as well asTrkB receptor protein expression in the hippocampus and PC regions suggesting that the deterioration of synaptic plasticity in LPS-treated rats may be mediated by dysfunctional BDNF-TrkB pathway (Fig. 4). Various previous studies have also reported that administration of LPS leads to significant reduction of BDNF mRNA and protein expression in hippocampus and different cortical regions resulting in depressive like sickness behavior and cognitive impairments in these animals [63, 64]. Interestingly, ASH-WEX pretreatment suppressed these LPS-induced changes in BDNF and TrkB receptor mRNA as well as protein expression thus indicating the potential activity of ASH-WEX in maintaining neuronal plasticity and recovery of functional deficits, working memory and learning.
BDNF binding causes tyrosine phosphorylation in the intracellular Trk domains which in turn triggers PLCγ-IP3 and PI3K-Akt intracellular cascades thereby initiating the transduction of BDNF signal [61, 65]. Downregulation in mRNA expression of PI3K and Akt and further phosphorylated form of Akt in LPS-treated rats may suggest suppression of cell survival signals which was restored with ASH-WEX supplementation. Phosphorylated Akt then promotes cell survival and inhibits apoptosis through inactivation of apoptotic protein BAD with corresponding activation of Bcl-xL thus protecting the cell from apoptotic stimuli. LPS-induced decrease in expression of Bcl-xL was also restored by ASH-WEX pretreatment thus indicating anti-apoptotic potential of ASH-WEX against inflammation associated neurotoxicity (Fig. 4). Parallel study using in vitro culture system clearly showed induction of neurotoxicity in neuronal cultures exposed to LPS stimulated microglial conditioned medium and microglial-neuronal co-cultures which may be explained by in vivo observation of suppression of PI3K-Akt signaling by LPS. ASH-WEX and FIV pretreatment to LPS-treated cultures may have maintained the neuronal health by regulating cell survival PI3K-Akt pathway. Hwang et al. (2017) also reported that aqueous methanolic extract of Ashwagandha roots and its active ingredients withaferin A, withanolide A protect the hippocampal neurons from nutritional stress by upregulating the expression of BDNF and PI3K-Akt baseline levels . Likewise in BDNF triggered PLCγ signaling cascade, ASH-WEX supplementation also restored PLCγ and IP3R mRNA expression which was downregulated by LPS (Fig. 4).
Infrastructure was provided by University Grants Commission (UGC), India under UPE and CPEPA schemes and Department of Biotechnology (DBT), India. MG is thankful to Council of Scientific and Industrial Research (CSIR), India for research fellowship. The authors are thankful to Shaffi Manchanda, Taranjeet Kaur, Anuradha Sharma, Harpal Singh and Manpreet Kaur for their kind support during execution of experiments.
Availability of materials
The datasets used and analyzed during the current study available from the corresponding author on reasonable request.
The study was planned and proposed by GK and MG. MG did all the experiments and analyzed the data. The manuscript was jointly written by MG and GK. Funding to carry out the work reported in the manuscript was provided by GK from her research grant. Both GK and MG reviewed and approved the final manuscript.
The current research work was funded by Department of Biotechnology (DBT), GOI to Prof. Gurcharan Kaur [Grant No: BT/PR12200/MED/30/1439/2014]. The funding agency had no role to design the study; collecting, analyzing and interpreting the data; in writing of manuscript and to select journal for submitting the article for publication.
Institutional Animal Ethical Committee, Guru Nanak Dev University, Amritsar, Punjab, India, which approved the experimental protocols is registered with “Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), GOI” (Registration No. 226/CPCSEA) (Permission No. 226/CPCSEA/2015/18). The experiments were performed in accordance with the relevant guidelines of ‘Animal Care and Use’ laid down by the same committee.
Consent for publication
The authors declare that they have no competing interests. As a member of the editorial board (Associate Editor) of BMC Complementary and Alternative Medicine (BCAM) Journal, Prof. Gurcharan Kaur hereby declare that she has no competing interest in this MS publication in BCAM.
- 8.Brown GC. Vilalta A (2015) How microglia kill neurons. Brain Res. 1628;2015:288–97.Google Scholar
- 14.Singh N, Bhalla M, de Jager P, Gilca M. An overview on ashwagandha: a Rasayana (rejuvenator) of Ayurveda. Afr J Tradit Complement Altern Med. 2011;8.Google Scholar
- 23.Kurapati KRV, Samikkannu T, Atluri VSR, Kaftanovskaya E, Yndart A, Nair MP. β-Amyloid1–42, HIV-1Ba-L (Clade B) infection and drugs of abuse induced degeneration in human neuronal cells and protective effects of Ashwagandha (Withania somnifera) and its constituent Withanolide A. PLoS One. 2014;(9):e112818.CrossRefGoogle Scholar
- 40.Yang HL, Huang PJ, Liu YR, Kumar KJS, Hsu LS, Lu TL, Chia YC, Takajo T, Kazuroni A, Hseu YC. Toona sinensis inhibits LPS-induced inflammation and migration in vascular smooth muscle cells via suppression of reactive oxygen species and NF-B signaling pathway. Oxidative Med Cell Longev. 2014;2014.Google Scholar
- 44.Mishra R, Manchanda S, Gupta M, Kaur T, Saini V, Sharma A, Kaur, G. (2016). Tinospora cordifolia ameliorates anxiety-like behavior and improves cognitive functions in acute sleep deprived rats. Sci rep. 2016;6:25564.Google Scholar
- 71.Tohda C, Komatsu K, Kuboyama T. Scientific basis for the anti-dementia drugs of constituents from Ashwagandha (Withania somnifera). J Tradit Med. 2005;22:176–82.Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.